Sandra A. Rempel

Controlling integrin specificity and stem cell differentiation in 2D and 3D environments through regulation of fibronectin domain stability

The extracellular matrix (ECM) exerts powerful control over many cellular phenomena, including stem cell differentiation. As such, design and modulation of ECM analogs to ligate specific integrin is a promising approach to control cellular processes in vitro and in vivo for regenerative medicine strategies. Although fibronectin (FN), a crucial ECM protein in tissue development and repair, and its RGD peptide are widely used for cell adhesion, the promiscuity with which they engage integrins leads to difficulty in control of receptor-specific interactions. Recent simulations of force-mediated unfolding of FN domains and sequences analysis of human versus mouse FN suggest that the structural stability of the FNs central cell-binding domains (FN III9-10) affects its integrin specificity. Through production of FN III9-10 variants with variable stabilities, we obtained ligands that present different specificities for the integrin alpha(5)beta(1) and that can be covalently linked into fibrin matrices. Here, we demonstrate the capacity of alpha(5)beta(1) integrin-specific engagement to influence human mesenchymal stem cell (MSC) behavior in 2D and 3D environments. Our data indicate that alpha(5)beta(1) has an important role in the control of MSC osteogenic differentiation. FN fragments with increased specificity for alpha(5)beta(1) versus alpha(v)beta(3) results in significantly enhanced osteogenic differentiation of MSCs in 2D and in a clinically relevant 3D fibrin matrix system, although attachment/spreading and proliferation were comparable with that on full-length FN. This work shows how integrin-dependant cellular interactions with the ECM can be engineered to control stem cell fate, within a system appropriate for both 3D cell culture and tissue engineering.

MicroRNA-145 Is Downregulated in Glial Tumors and Regulates Glioma Cell Migration by Targeting Connective Tissue Growth Factor

Glioblastomas (GBM), the most common and aggressive type of malignant glioma, are characterized by increased invasion into the surrounding brain tissues. Despite intensive therapeutic strategies, the median survival of GBM patients has remained dismal over the last decades. In this study we examined the expression of miR-145 in glial tumors and its function in glioma cells. Using TCGA analysis and real-time PCR we found that the expression of miR-145/143 cluster was downregulated in astrocytic tumors compared to normal brain specimens and in glioma cells and glioma stem cells (GSCs) compared to normal astrocytes and neural stem cells. Moreover, the low expression of both miR-145 and miR-143 in GBM was correlated with poor patient prognosis. Transfection of glioma cells with miR-145 mimic or transduction with a lentivirus vector expressing pre-miR 145 significantly decreased the migration and invasion of glioma cells. We identified connective tissue growth factor (CTGF) as a novel target of miR-145 in glioma cells; transfection of the cells with this miRNA decreased the expression of CTGF as determined by Western blot analysis and the expression of its 3′-UTR fused to luciferase. Overexpression of a CTGF plasmid lacking the 3′-UTR and administration of recombinant CTGF protein abrogated the inhibitory effect of miR-145 on glioma cell migration. Similarly, we found that silencing of CTGF decreased the migration of glioma cells. CTGF silencing also decreased the expression of SPARC, phospho-FAK and FAK and overexpression of SPARC abrogated the inhibitory effect of CTGF silencing on cell migration. These results demonstrate that miR-145 is downregulated in glial tumors and its low expression in GBM predicts poor patient prognosis. In addition miR-145 regulates glioma cell migration by targeting CTGF which downregulates SPARC expression. Therefore, miR-145 is an attractive therapeutic target for anti-invasive treatment of astrocytic tumors.

SPARC-induced increase in glioma matrix and decrease in vascularity are associated with reduced VEGF expression and secretion.

Glioblastomas are heterogeneous tumors displaying regions of necrosis, proliferation, angiogenesis, apoptosis and invasion. SPARC, a matricellular protein that negatively regulates angiogenesis and cell proliferation, but enhances cell deadhesion from matrix, is upregulated in gliomas (Grades II–IV). We previously demonstrated that SPARC promotes invasion while concomitantly decreasing tumor growth, in part by decreasing proliferation of the tumor cells. In other cancer types, SPARC has been shown to influence tumor growth by altering matrix production, and by decreasing angiogenesis via interfering with the VEGF‐VEGFR1 signaling pathway. We therefore examined whether the SPARC‐induced decrease in glioma tumor growth was also, in part, due to alterations in matrix and/or decreased vascularity, and assessed SPARC‐VEGF interactions. The data demonstrate that SPARC upregulates glioma matrix, collagen I is a constituent of the matrix and SPARC promotes collagen fibrillogenesis. Furthermore, SPARC suppressed glioma vascularity, and this was accompanied by decreased VEGF expression and secretion, which was, in part, due to reduced VEGF165 transcript abundance. These data indicate that SPARC modulates glioma growth by altering the tumor microenvironment and by suppressing tumor vascularity through suppression of VEGF expression and secretion. These experiments implicate a novel mechanism, whereby SPARC regulates VEGF function by limiting the available growth factor. Because SPARC is considered to be a therapeutic target for gliomas, a further understanding of its complex signaling mechanisms is important, as targeting SPARC to decrease invasion could undesirably lead to the growth of more vascular and proliferative tumors.

HSP27 mediates SPARC-induced changes in glioma morphology, migration, and invasion

Secreted protein acidic and rich in cysteine (SPARC) regulates cell–extracellular matrix interactions that influence cell adhesion and migration. We have demonstrated that SPARC is highly expressed in human gliomas, and it promotes brain tumor invasion in vitro and in vivo. To further our understanding regarding SPARC function in glioma migration, we transfected SPARC‐green fluorescent protein (GFP) and control GFP vectors into U87MG cells, and assessed the effects of SPARC on cell morphology, migration, and invasion after 24 h. The expression of SPARC was associated with elongated cell morphology, and increased migration and invasion. The effects of SPARC on downstream signaling were assessed from 0 to 6 h and 24 h. SPARC increased the levels of total and phosphorylated HSP27; the latter was preceded by activation of p38 MAPK and inhibited by the p38 MAPK inhibitor SB203580. Augmented expression of SPARC was correlated with increased levels of HSP27 mRNA. In a panel of glioma cell lines, increasing levels of SPARC correlated with increasing total and phosphorylated HSP27. SPARC and HSP27 were colocalized to invading cells in vivo. Inhibition of HSP27 mRNA reversed the SPARC‐induced changes in cell morphology, migration, and invasion in vitro. These data indicate that HSP27, a protein that regulates actin polymerization, cell contraction, and migration, is a novel downstream effector of SPARC‐regulated cell morphology and migration. As such, it is a potential therapeutic target to inhibit SPARC‐induced glioma invasion.

Hippocampal connexin 43 expression in human complex partial seizure disorder

An increase in the cellular production of gap junction proteins and increased numbers of gap junctions in the neuronoglial syncytium of an epileptic focus have been proposed as a possible mechanism underlying synchronization of discharge. To study this issue, both Northern and Western blot analyses of the gap junction protein connexin 43 mRNA and protein abundance were performed on hippocampal tissue resected from patients presenting with a complex partial seizure disorder arising from the medial temporal area and the hippocampus in particular. Samples from 15 patients with medically intractable seizures were compared to those from 5 nonepileptic patients requiring temporal lobectomy in life-threatening situations. Six of the 15 epileptic patients underwent noninvasive electrographic recording, whereas the remaining 9 patients required intracerebral electrodes for extraoperative recording and therefore showed a more discrete focality than the noninvasive recordings. A decline in the mean levels of connexin 43 mRNA expressed predominantly in astrocytes was noted in the epileptic patient groups, particularly for those cases requiring intracranial electrode placement where ictal onset was more clearly established to be intrahippocampal. Quantitation of connexin 43 protein in both epileptogenic and nonepileptogenic hippocampal tissues showed no significant differences in expression. Although mean values for mRNA showed a decline, clinical outcomes postoperatively showed no correlation with either mRNA or protein expression individually in our epileptic population. The findings indicate that there is effectively no upregulation of mRNA and no increased production of connexin 43 protein in response to the development of epileptogenicity. Rather it appears the influence of gap junctions as a substrate of epileptogenicity in any mechanism(s) underlying synchrony or electrical propagation may be a function of the dynamic state (open versus closed) of the membrane-bound gap junction.

PTEN augments SPARC suppression of proliferation and inhibits SPARC-induced migration by suppressing SHC-RAF-ERK and AKT signaling

SPARC (secreted protein acidic and rich in cysteine) is expressed in all grades of astrocytoma, including glioblastoma (GBM). SPARC suppresses glioma growth but promotes migration and invasion by mediating integrin and growth factor receptor-regulated kinases and their downstream effectors. PTEN (phosphatase and tensin homolog deleted on chromosome 10), which is commonly lost in primary GBMs, negatively regulates proliferation and migration by inhibiting some of the same SPARC-mediated signaling pathways. This study determined whether PTEN reconstitution in PTEN-mutant, SPARC-expressing U87MG cells could further suppress proliferation and tumor growth but inhibit migration and invasion in SPARC-expressing cells in vitro and in vivo, and thereby prolong survival in animals with xenograft tumors. In vitro, PTEN reduced proliferation and migration in both SPARC-expressing and control cells, with a greater suppression in SPARC-expressing cells. PTEN reconstitution suppressed AKT activation in SPARC-expressing and control cells but suppressed the SHC-RAF-ERK signaling pathway only in SPARC-expressing cells. Importantly, coexpression of SPARC and PTEN resulted in the smallest, least proliferative tumors with reduced invasive capacity and longer animal survival. Furthermore, direct inhibition of the AKT and SHC-RAF-ERK signaling pathways suppressed the proliferation and migration of SPARC-expressing cells in vitro. These findings demonstrate that PTEN reconstitution or inhibition of signaling pathways that are activated by the loss of PTEN provide potential therapeutic strategies to inhibit SPARC-induced invasion while enhancing the negative effect of SPARC on tumor growth.

Inhibition of HSP27 alone or in combination with pAKT inhibition as therapeutic approaches to target SPARC-induced glioma cell survival

BackgroundThe current treatment regimen for glioma patients is surgery, followed by radiation therapy plus temozolomide (TMZ), followed by 6 months of adjuvant TMZ. Despite this aggressive treatment regimen, the overall survival of all surgically treated GBM patients remains dismal, and additional or different therapies are required. Depending on the cancer type, SPARC has been proposed both as a therapeutic target and as a therapeutic agent. In glioma, SPARC promotes invasion via upregulation of the p38 MAPK/MAPKAPK2/HSP27 signaling pathway, and promotes tumor cell survival by upregulating pAKT. As HSP27 and AKT interact to regulate the activity of each other, we determined whether inhibition of HSP27 was better than targeting SPARC as a therapeutic approach to inhibit both SPARC-induced glioma cell invasion and survival.ResultsOur studies found the following. 1) SPARC increases the expression of tumor cell pro-survival and pro-death protein signaling in balance, and, as a net result, tumor cell survival remains unchanged. 2) Suppressing SPARC increases tumor cell survival, indicating it is not a good therapeutic target. 3) Suppressing HSP27 decreases tumor cell survival in all gliomas, but is more effective in SPARC-expressing tumor cells due to the removal of HSP27 inhibition of SPARC-induced pro-apoptotic signaling. 4) Suppressing total AKT1/2 paradoxically enhanced tumor cell survival, indicating that AKT1 or 2 are poor therapeutic targets. 5) However, inhibiting pAKT suppresses tumor cell survival. 6) Inhibiting both HSP27 and pAKT synergistically decreases tumor cell survival. 7) There appears to be a complex feedback system between SPARC, HSP27, and AKT. 8) This interaction is likely influenced by PTEN status. With respect to chemosensitization, we found the following. 1) SPARC enhances pro-apoptotic signaling in cells exposed to TMZ. 2) Despite this enhanced signaling, SPARC protects cells against TMZ. 3) This protection can be reduced by inhibiting pAKT. 4) Combined inhibition of HSP27 and pAKT is more effective than TMZ treatment alone.ConclusionsWe conclude that inhibition of HSP27 alone, or in combination with pAKT inhibitor IV, may be an effective therapeutic approach to inhibit SPARC-induced glioma cell invasion and survival in SPARC-positive/PTEN-wildtype and SPARC-positive/PTEN-null tumors, respectively.

Prognostic significance of telomere maintenance mechanisms in pediatric high-grade gliomas

Children with high-grade glioma, including diffuse intrinsic pontine glioma (DIPG), have a poor prognosis despite multimodal therapy. Identifying novel therapeutic targets is critical to improve their outcome. We evaluated prognostic roles of telomere maintenance mechanisms in children with HGG, including DIPG. A multi-institutional retrospective study was conducted involving 50 flash-frozen HGG (35 non-brainstem; 15 DIPG) tumors from 45 children (30 non-brainstem; 15 DIPG). Telomerase activity, expression of hTERT mRNA (encoding telomerase catalytic component) and TERC (telomerase RNA template) and alternative lengthening of telomeres (ALT) mechanism were assayed. Cox Proportional Hazard regression analyses assessed association of clinical and pathological variables, TERC and hTERT levels, telomerase activity, and ALT use with progression-free or overall survival (OS). High TERC and hTERT expression was detected in 13/28 non-brainstem HGG samples as compared to non-neoplastic controls. High TERC and hTERT expression was identified in 13/15 and 11/15 DIPG samples, respectively, compared to controls. Evidence of ALT was noted in 3/11 DIPG and 10/19 non-brainstem HGG specimens. ALT and telomerase use were identified in 4/19 non-brainstem HGG and 2/11 DIPG specimens. In multivariable analyses, increased TERC and hTERT levels were associated with worse OS in patients with non-brainstem HGG, after controlling for tumor grade or resection extent. Children with HGG and DIPG, have increased hTERT and TERC expression. In children with non-brainstem HGG, increased TERC and hTERT expression levels are associated with a worse OS, making telomerase a promising potential therapeutic target in pediatric HGG.

Proteolysis of the matricellular protein hevin by matrix metalloproteinase‐3 produces a SPARC‐like fragment (SLF) associated with neovasculature in a murine glioma model

The matricellular SPARC‐family member hevin (Sparc‐like 1/SPARCL‐1/SC1/Mast9) contributes to neural development and alters tumor progression in a range of mammalian models. Based on sequence similarity, we hypothesized that proteolytic digestion of hevin would result in SPARC‐like fragments (SLF) that affect the activity and/or location of these proteins. Incubation of hevin with matrix metalloproteinase‐3 (MMP‐3), a protease known to cleave SPARC, produced a limited number of peptides. Sequencing revealed the major proteolytic products to be SPARC‐like in primary structure. In gliomas implanted into murine brain, a SLF was associated with SPARC in the neovasculature but not with hevin, the latter prominent in the astrocytes encompassed by infiltrating tumor. In this model of invasive glioma that involves MMP‐3 activity, host‐derived SLF was not observed in the extracellular matrix adjacent to tumor cells. In contrast, it occurred with its homolog SPARC in the angiogenic response to the tumor. We conclude that MMP‐3‐derived SLF is a marker of neovessels in glioma, where it could influence the activity of SPARC. J. Cell. Biochem. 112: 3093–3102, 2011.

MOLECULAR BIOLOGY OF NERVOUS SYSTEM TUMORS

Many genetic alterations that contribute to CNS tumorigenesis and progression have been identified. One goal of such studies is to identify loci that would serve as diagnostic prognostic markers or both. A significant advance is the observation that chromosome 1p loss identified anaplastic oligodendroglioma and a subset of high-grade glioma patients who responded to chemotherapy and had longer survival times. Combined 1p and 19q loss was a predictor of prolonged survival of patients having pure oligodendrogliomas. Such markers eventually may be used to identify patients upfront who would benefit from treatment, while sparing patients who would not benefit. Although many molecular participants involved in the biologic pathways that promote proliferation, angiogenesis, and invasion have been elucidated, there are still many gaps in clinicians knowledge. It is expected that the use of the human genome project information and databases such as SAGEmap, in combination with techniques such as cDNA arrays and proteomics, will facilitate greatly the identification of novel genes that contribute to CNS tumors. cDNA arrays and tissue arrays will permit the construction of CNS-specific screening tools that will permit the identification of tumor-specific mutations and alterations so that patient-specific therapies can be designed.